Nitrate
contamination of groundwater is a growing concern worldwide. Considering that
analytical methods have changed over the past forty years, data collected using
1950's techniques has been incorrectly compared to data collected today. Without
applying random and bias errors associated with each independent method, these
reported changes in NO3-N concentrations are scientifically invalid.
This study was initiated to determine the errors associated with analytical
procedure, seasonal sampling, and storage method for well water NO3-N
analysis. Nitrate-N concentrations for fifty water wells in Grant, Garfield, and
Kingfisher counties, obtained during the period 1950-1972, were acquired through
cooperation with The United States Geological Survey and The Oklahoma Water
Resources Board. Well water samples were taken at different dates from these
same locations in 1993 and 1994. For each sampling date, four samples were taken
from each well whereby two were frozen immediately (as per EPA protocol), and
two were stored at ambient temperature for five to ten days as was common in
1950. Water samples were then analyzed using phenoldisulfonic acid (1950) and
automated cadmium reduction(1993) procedures. Mean NO3-N values from
analysis using cadmium reduction were significantly higher than values obtained
using the phenoldisulfonic acid procedure, reflecting the bias associated with
making direct numerical comparisons between procedures. Comparisons made between
1950 and 1993 well water analysis (employing the same phenoldisulfonic acid
procedure) indicate that few significant increases have taken place over the
past forty years. No relationship between well water NO3-N and depth
to aquifer was found which indirectly suggests that NO3-N leaching
was not significant for this population of 50 wells.

Introduction

Nitrate-N
contamination of groundwater is a growing concern worldwide. When
nitrate-nitrogen(NO3-N) is ingested by infants, nitrate can be
reduced to nitrite. Nitrite-N can occupy sites on hemoglobin that would normally
carry oxygen. This reduced oxygen carrying capacity of the blood is what leads
to methemoglobinemia, or "blue baby" syndrome which can be fatal to infants
under six months of age. This condition is, however, treatable and fatalities
are rare. In the last thirty years, only one death in the United States was
linked to Nitrate poisoning caused from drinking well water (Fedkiw, 1991). The
maximum level for NO3-N in drinking water is 10 mg/kg(as set by the
Environmental Protection Agency). Results from a national survey (National
Pesticide Survey, 1990) noted that only a small percentage (2.4%) of rural water
wells exceed this level.

The issue
that groundwater NO3-N concentrations have increased over time is not
disputed. Nitrate-N occurs naturally in the soil (Johnson, 1993). Despite this
fact, agricultural chemicals, particularly N fertilizers, have received the bulk
of the blame for the increase. A computer based literature search performed by
L.W. Canter found 34 references where groundwater NO3-N was
associated with fertilizer use (Canter, 1987). In order for fertilizers to be
directly responsible for groundwater contamination, NO3-N would have
to be leached out of the soil profile. The occurrence of this is preceded by
other processes resulting in much higher levels of N removal (Mills et al.,
1974; Sharpe et al., 1988; Hooker et al., 1980; Aulakh et al., 1984). DeWalle
and Schaff, 1980, Hallberg, 1983, and Olsen ,1974, among many others, have
reported agriculture related increases in groundwater NO3-N
concentrations over time periods of up to thirty years. Considering that
analytical methods have changed since the 1950's, direct numerical comparison
between data obtained thirty years ago and data obtained today is erroneous.
Schepers et al., 1991, Walters and Malzer, 1990, and Webster et al., 1986, have
also evaluated the impacts of N fertilizers without addressing the analytical
errors associated with these estimates. Without adequately assessing the random
and bias errors associated with an independent estimate, researchers are at risk
of making scientifically invalid conclusions about changes in NO3-N
concentrations.

The
objective of this research was to determine the errors associated with
analytical procedure, seasonal sampling, and storage method for well water NO3-N
analysis. A thorough understanding of the errors associated with estimates of NO3-N
in groundwater will assist researchers in identifying where significant
increases have taken place.

Materials and Methods

Fifty
water wells in Garfield, Grant, and Kingfisher counties (Figure 1) were
selected for comprehensive sampling. These wells were selected on the basis that
NO3-N data collected during the time period 1953-1972 was available
(Bingham and Bergman, 1980; Dover, 1953; U.S.G.S., 1993). These counties also
have substantial agricultural activity associated with continuous wheat
production and N fertilization. Locations and physical characteristics for each
well site are found in Table 1. Tax records obtained from the three counties
were used to determine current ownership of the property on which each well was
located. The owners were contacted and informed about the experiment. Permission
was obtained from the well owners to begin quarterly well water sampling
beginning in the fall of 1993.

In order
to obtain a representative groundwater sample, it is desirable to take the
sample directly from the aquifer. However, 39 of the 50 wells contain in-place
semi-permanent mounted pumps which limit the options available for groundwater
sampling. These wells were pumped for 5-10 minutes prior to sampling so that
water in the well reasonably represented that of the aquifer. Of the 11 other
wells, 7 were collected via windmills and 4 were collected using a teflon
bailer. All samples were taken using proper sampling protocol (Barcelona et al.,
1987; Davis et al., 1993; Scalf et al., 1981). For each sampling date, four
samples were taken from each well whereby two were frozen immediately (AOAC,
1984) by being placed in a cooler containing dry ice and two were stored at
ambient temperatures for five to ten days as was common in 1950.

Frozen
and non-frozen samples were analyzed using two methods. One method was
phenoldisulfonic acid (Bremner, 1965; Chapman and Pratt, 1961; Snell, 1949), a
procedure commonly used in 1950 when the original NO3-N data was
collected. The other method, automated cadmium reduction (Henriksen and
Selmer-Olsen, 1970; Jackson et al., 1975), was performed using the
Lachat-Quickchem automated flow injection system (1993). Statistical analysis
of data was performed using procedures outlined by the SAS institute (SAS,
1988). Geographic Information Systems was used to produce surface and contour
maps showing relationships among wells and analytical methods.

Results and Discussion

Significant differences (.0001 probability) in well water NO3-N were
found between season of sampling and method of analysis. The mean NO3-N
value for samples taken in each of the four seasons ranged from 6.97 ± 5.8 in
the fall to 9.59 ± 8.65 in the summer. This agrees with work done by Gilliam et
al., 1974, Hallberg et al., 1983, and McDonald and Splinter, 1982, all of whom
found NO3-N levels for a specific well to vary among seasons. This
reflects a bias error associated with seasonal sampling. The difference between
frozen and non-frozen samples was statistically insignificant (.8415
probability). The largest difference in means was found when comparisons were
made between analytical methods. Mean well water NO3-N concentrations
found by using the phenoldisulfonic acid procedure were 3.2 mg/kg lower than
those found using cadmium reduction (9.4 ± 7.5 vs 6.2 ± 4.7). Possible
explanations for this difference are that a loss of NO3-N occurred
when using the phenoldisulfonic acid procedure, or that the accuracy of
phenoldisulfonic acid is poor in terms of NO3-N detection. In either
case, an error exists. Comparing population means generated by all possible
combinations of the variables (season, storage, and method) resulted in
overlapping standard deviations which seriously restricted whether or not
conclusive changes had taken place when comparing data obtained using
independent methods (Figure 2). Comparisons made between 1950 and 1993 well
water analysis (employing the same phenoldisulfonic acid procedure) indicated
that 57 % of all wells sampled have shown either a significant decrease or no
significant change at all over the past forty years (Table 2).

No
relationship was found between the average depth to aquifer and well water NO3-N
in either 1953, 1993 or the difference (1993-1953, Figure 3). Because
fertilizer use was not common before 1950, increased fertilizer use from
1950-present should have resulted in significant well water NO3-N
increases in more shallow aquifers if in fact NO3-N leaching from
surface applied fertilizers was present. If NO3-N leaching from the
excessive use of N fertilizers were to have been significant over this forty
year time period, depth to the aquifer (on these extremely shallow wells, <30
ft) and well water NO3-N should have been negatively correlated.

Table 1. Location, soil type,
major land use and average depth to aquifer for each well sampled in 1953 and
1993, Grant, Garfield and Kingfisher Counties, OK.

WELL

COUNTY

LOCATION

SOIL
TYPE

MAJOR
LAND USE

AVG.
DEPTH TO AQUIFER

IN FEET
BELOW SURFACE

1

KINGFISHER

15N-07W-02 CC

KINGFISHER SILT LOAM, 3 TO 5 % SLOPE

CROPLAND

10

2

KINGFISHER

16N-07W-29 C

RENFROW
CLAY LOAM, 1 TO 3 % SLOPE

CROPLAND

9

3

KINGFISHER

17N-07W-36 B

NORGE
FINE SANDY LOAM, 0 TO 1 % SLOPE

CROPLAND

19

4

KINGFISHER

17N-06W-31 B

PORT
SILT LOAM, 0 TO 1 % SLOPE

RANGELAND

19

5

KINGFISHER

17N-05W-31 D

KINGFISHER SILT LOAM, 3 TO 5 % SLOPE

CROPLAND

35

6

KINGFISHER

17N-05W-32 B

PORT
CLAY LOAM, 0 TO 1 % SLOPE

CROPLAND

35

7

KINGFISHER

17N
-05W-32 C

PORT
CLAY LOAM, 0 TO 1 % SLOPE

CROPLAND

35

8

KINGFISHER

17N-05W-35 D

PRATT
LOAMY FINE SAND, UNDULATING

CROPLAND

22

9

KINGFISHER

17N-05W-36 A

PRATT
LOAMY FINE SAND, UNDULATING

CROPLAND

27

10

KINGFISHER

17N-05W-18 D

PRATT
LOAMY FINE SAND, HUMMOCKY

CROPLAND

25

11

KINGFISHER

17N-07W-11 D

LINCOLN
LOAMY FINE SAND

IRRIGATED PASTURELAND

10

12

KINGFISHER

17N-08W-4 C

NORGE-SLICKSPOT COMPLEX, 1 TO 3 % SLOPE

CROPLAND

7

13

KINGFISHER

17N-07W-06 B

NORGE
FINE SANDY LOAM, 0 TO 1 SLOPE

CROPLAND

5

14

KINGFISHER

17N-07W-01 C

PRATT
LOAMY FINE SAND, HUMMOCKY

RANGELAND

7

15

KINGFISHER

18N-06W-32 B

PRATT
LOAMY FINE SAND, UNDULATING

CROPLAND

28

16

KINGFISHER

18N-07W-28 C

PRATT
LOAMY FINE SAND, UNDULATING

CROPLAND

11

17

KINGFISHER

18N-07W-24 A

DOUGHERTY-EUFALA LOAMY FINE SAND, HUMMOCKY

IRRIGATED CROPLAND

29

18

KINGFISHER

18N-07W-13 AC

CARWILE
LOAMY FINE SAND

CROPLAND

25

19

KINGFISHER

18N-07W-15 A

SHELLABARGER FINE SANDY LOAM, 5 TO 8 % SLOPE

RANGELAND

26

20

KINGFISHER

18N-07W-08 D

DOUGHERTY-EUFALA LOAMY FINE SAND, UNDULATING

CROPLAND

26

21

KINGFISHER

18N-08W-02 D

PRATT
LOAMY FINE SAND, HUMMOCKY

CROPLAND

18

22

KINGFISHER

18N-O8W-02 C

DOUGHERTY-EUFALA LOAMY FINE SAND, HUMMOCKY

PASTURELAND

18

23

KINGFISHER

19N-07W-29 D

DOUGHERTY-EUFALA LOAMY FINE SAND, UNDULATING

CROPLAND

33

24

KINGFISHER

19N-07W-8 D

CARWILE
LOAMY FINE SAND

CROPLAND

6

25

KINGFISHER

19N-08W-11 DA

DOUGHERTY-EUFALA LOAMY FINE SAND, HUMMOCKY

IRRIGATED PASTURELAND

13

26

KINGFISHER

19N-08W-11 A

DOUGHERTY-EUFALA LOAMY FINE SAND, HUMMOCKY

IRRIGATED PASTURELAND

13

27

KINGFISHER

15N-07W-02 C

ALLUVIAL
AND BROKEN LAND

CROPLAND

7

28

KINGFISHER

19N-09W-23 BBB

EUFALA
FINE SAND

CROPLAND

11

29

KING
FISHER

19N-09W-10 AB

PRATT
LOAMY FINE SAND, UNDULATING

RANGELAND

17

30

KINGFISHER

19N-08W-05 B

DOUGHERTY-EUFALA LOAMY FINE SAND, UNDULATING

CROPLAND

8

31

GARFIELD

20N-08W-23 CC

SHELLABARGER FINE SANDY LOAM, 1 TO 3 % SLOPE

CROPLAND

20

32

GARFIELD

20N-05W-28 A

PORT
CLAY LOAM

CROPLAND

18

33

GARFIELD

20N-03-W-26 BBC

ERODED
CLAYEY LAND

RANGELAND

38

34

GARFIELD

21N-04W-11 D

KIRKLAND
SILT LOAM, 0 TO 1 % SLOPE

CROPLAND

19

35

GARFIELD

21N-06W-12 CCB

PORT
SILT LOAM, 0 TO 1 % SLOPE

CROPLAND

18

36

GARFIELD

21N-07W-23 BBC

POND
CREEK SILT LOAM, 0 TO 1 % SLOPE

CROPLAND

18

37

GARFIELD

21N-08W-20 CCC

DRUMMOND
SOILS

RANGELAND

11

38

GARFIELD

22N-O8W-18 A

GRANT-NASH SILT LOAM, 5 TO 8 % SLOPE

RANGELAND

3

39

GARFIELD

22N-07W-01 D

SHELLABARGER-CARWILE FINE SANDY LOAM, UNDULATING

URBAN/BUILT UP LAND

18

41

GARFIELD

24N-03W-05 B

RENFROW-VERNON COMPLEX, 3 TO 5 % SLOPE, ERODED

CROPLAND

10

42

GARFIELD

24N-05W-03 C

KIRKLAND-RENFROW SILT LOAM, 1 TO 3 % SLOPE

CROPLAND

18

43

GARFIELD

24N-05W-35 A

KIRKLAND
SILT LOAM, 0 TO 1 % SLOPE

CROPLAND

17

44

GARFIELD

23N-06W-7 D

MENO
LOAMY FINE SAND, UNDULATING

RANGELAND

20

45

GARFIELD

23N-07W-7 C

POND
CREEK SILT LOAM, 0 TO 1 % SLOPE

CROPLAND

27

46

GARFIELD

24N-07W-21 ADD

GRANT
SILT LOAM, 3 TO 5 % SLOPE, ERODED

CROPLAND

18

48

GRANT

25N-05W-6 D

YAHOLA
FINE SANDY LOAM, OCCASIONALLY FLOODED

RANGELAND

10

49

GRANT

28N-07W-24 A

QUINLAN-WOODWARD LOAM, 3 TO 12 % SLOPE

RANGELAND

6

50

GRANT

27N-03W-28 B

RENFROW
SILTY CLAY LOAM, 2 TO 5 % SLOPE, ERODED

CROPLAND

26

51

GRANT

27N-03W-22 B

McLAIN-DRUMMOND
SILT LOAM, RARELY FLOODED

RANGELAND

30

52

GRANT

27N-03W-23 A

KIRKLAND
SILT LOAM, 1 TO 3 % SLOPE

CROPLAND

30

Table 2.
Nitrate-N concentrations for each well sampled between 1950 and 1972 and the
1993 sampling data analyzed using the phenoldisulfonic acid procedure when
samples were not frozen, Grant, Garfield and Kingfisher Counties, OK.